MOLECULAR MECHANISMS OF TRAF3- DEFICIENT B LYMPHOMAGENESIS by Shanique Katrice Elaine Edwards A Dissertation submitted to the Graduate School-New Brunswick Rutgers, the State University of New Jersey and The Graduate School of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Program in Cell and Developmental Biology written under the direction of Ping Xie and approved by ________________________ ________________________ ________________________ ________________________ New Brunswick, New Jersey May 2015 ABSTRACT OF THE DISSERTATION Molecular Mechanisms of TRAF3-deficient B Lymphomagenesis By SHANIQUE KATRICE ELAINE EDWARDS Dissertation Director: Ping Xie B cell neoplasms, including leukemias, lymphomas and myelomas, are a common type of cancer, but they remain difficult to treat. This outlines a need for a better understanding of the mechanisms by which malignant transformation occurs, in order to come up with better therapeutic strategies. Recently, TRAF3 has been shown to act as a tumor suppressor, as mice with this gene specifically deleted in B cells develop B lymphomas. TRAF3 deletion causes prolonged B cell survival, allowing other secondary oncogenic alterations to occur. To elucidate these secondary alterations, we performed microarray analyses to identify genes which are differentially expressed in mouse B lymphomas. Two such genes that I have investigated in my thesis research are MCC and Sox5, both of which are significantly upregulated specifically in malignant B cells. MCC, mutated in colorectal cancer, has been previously identified as a tumor suppressor in colorectal cancer. We discovered that in malignant B cells, MCC acts as an ii oncogene to promote B cell survival and proliferation by modulating the signaling network centered at PARP1 and PHB1/2. The Sox5 gene encodes a transcription factor. Interestingly, we found that the Sox5 expressed in TRAF3-/- mouse B lymphomas represents a novel isoform of Sox 5, Sox5-BLM, which regulates malignant B cell proliferation by affecting the expression of p27 and -catenin. These two genes have the potential to be used as diagnostic markers and therapeutic targets in B cell malignancies. Based on the understanding of TRAF3 signaling mechanisms, we also conducted translational studies using anti-cancer drugs to manipulate TRAF3 downstream signaling components. We have tested drugs targeting NF-B2 and PKC, and found that oridonin and AD198 exhibit potent anti-tumor activities on B cell neoplasms with TRAF3 deletions or mutations. Furthermore, oridonin or AD 198 drastically potentiated the anti-cancer effects of bortezomib, an effective clinical drug for multiple myeloma. Taken together, our studies have gained new insights into the mechanisms underlying TRAF3 inactivation-initiated B lymphomagenesis, and have discovered novel therapeutic targets for B cell neoplasms. Our findings also provide a rationale for clinical evaluation of several drugs or drug combinations in the treatment of B cell malignancies. iii DEDICATION & ACKNOWLEDGEMENTS DEDICATION Sola Gratia, Soli Deo Gloria To my family, especially Dr. Daddy, Mommy, Mika, Shaq and Sadé: you have been my support through everything! There aren’t enough words to say how much you all mean to me. I hope that I’m at least half as great a woman as you have believed me to be. Love wunna bad! To my friends who have been there through it all, you are the best! Shamika, my frister, your realness has kept me from quitting many a time. Karlyn, thank you for your encouragement and commiseration through dissertation writing, and for your advice and our conversations. Ayodele Grace, your quiet but constant support has been everything to me. Javonni, your love and friendship have kept me sane throughout grad school. Ann, your excitement and energy have helped me to push through the tough days. I am also very thankful for all those who have supported and encouraged me over these five years: James Street, GCF, NBUMC, LGC, and my IC fam, if you hadn’t believed in me, I might not have had the courage to believe in myself. ACKNOWLEDGEMENTS MY ADVISER & COMMITTEE Many thanks to my dissertation adviser Dr. Ping Xie for her guidance and support over these years. I have learned so much from you about being a scientist and researcher. Thank you Dr. Lori Covey for your mentorship, support, guidance, encouragement, advice and listening ear. Thank you to Drs. Lisa Denzin, Bonnie Firestein & Ron Hart for pushing me to work harder, think more and be a better scientist. XIE LAB Thank you to everyone for the work that you have done to help me through this process, especially Yan, Carissa, Sukhdeep, Jackie, Anand, Ben and Punit. Without your help with experiments, I wouldn’t be here! Thank you to Almin, my lab mate for being there to bounce ideas off of, and to keep me on track. DIVISION OF LIFE SCIENCES & MOLECULAR BIOSCIENCES Thank you to the Division of Life Sciences for three wonderful years of financial support through the Teaching Assistant program! It has been such a pleasure working with Jana and everyone! Thanks also to the Molecular Biosciences Graduate Program for my first year, and summer support. iv TABLE OF CONTENTS Abstract of the dissertation ------------------------------------------------------------------------------------------- ii Dedication & Acknowledgements ---------------------------------------------------------------------------------- iv Dedication ------------------------------------------------------------------------------------------------------------ iv Acknowledgements ------------------------------------------------------------------------------------------------- iv Table of Contents ------------------------------------------------------------------------------------------------------- v List of Figures ------------------------------------------------------------------------------------------------------------ x List of Tables ----------------------------------------------------------------------------------------------------------- xii Introduction -------------------------------------------------------------------------------------------------------------- 1 B Cell Cancers: Prevalence & Unique Challenges --------------------------------------------------------- 1 TRAF Proteins and Immune Signaling ------------------------------------------------------------------------ 2 TRAF3, a Novel Tumor Suppressor ---------------------------------------------------------------------------- 4 TRAF3 Signaling Pathways in B lymphocytes --------------------------------------------------------------- 5 My Dissertation Research --------------------------------------------------------------------------------------- 10 Chapter 1 Mutated in colorectal cancer (MCC) is a novel oncogene in B lymphocytes ------------- 13 Abstract -------------------------------------------------------------------------------------------------------------- 13 1.1 Background----------------------------------------------------------------------------------------------------- 14 1.2 Materials and Methods -------------------------------------------------------------------------------------- 16 1.2.1 Mice, cell lines, and reagents ------------------------------------------------------------------------- 16 1.2.2 Transcriptome microarray analysis ----------------------------------------------------------------- 17 1.2.3 Taqman assays of the transcript expression of identified genes ---------------------------- 18 1.2.4 Splenic B cell purification and stimulation --------------------------------------------------------- 18 1.2.5 Taqman copy number assay of the mouse MCC gene ---------------------------------------- 19 1.2.6 Chromatin immunoprecipitation assay ------------------------------------------------------------- 19 1.2.7 Cloning of the full-length cDNA of the MCC gene from TRAF3-/- mouse B lymphomas and human MM cell lines ------------------------------------------------------------------------------------- 20 1.2.8 Generation of lentiviral MCC expression and shRNA vectors -------------------------------- 21 v 1.2.9 Lentiviral packaging and transduction of human MM cells ------------------------------------ 22 1.2.10 Growth curve determination, annexin V staining of apoptotic cells, cell cycle distribution and cell proliferation analyses ---------------------------------------------------------------- 22 1.2.11 Total protein lysates, fractionation of cytosol, mitochondria and microsomes (rich in ER), and immunoblot analysis ------------------------------------------------------------------------------- 23 1.2.12 Co-immunoprecipitation assay in whole cell lysates ------------------------------------------ 24 1.2.13 Co-immunoprecipitation assay in mitochondrial lysates-------------------------------------- 25 1.2.14 Mass spectrometry based-sequencing ----------------------------------------------------------- 25 1.2.15 Statistics ------------------------------------------------------------------------------------------------- 27 1.3 Results ---------------------------------------------------------------------------------------------------------- 27 1.3.1 Transcriptome microarray analysis of TRAF3 /- mouse B lymphomas --------------------- 27 1.3.2 Striking up-regulation of MCC in TRAF3 /- mouse B lymphomas but not in premalignant TRAF3 /- B lymphocytes -------------------------------------------------------------------------------------- 30 1.3.3 Aberrant up-regulation of MCC in human patient-derived